There is presently a dearth of candidate HIV vaccines that are considered suitable for wide-scale testing in humans, particularly when considering vaccines capable of inducing protective humoral immunity. Whereas live, attenuated viruses may provide protection against more pathogenic strains, safety considerations are likely to preclude the widespread use of such vaccines. A difficulty with purified envelope subunit vaccines is that while the best of these have been able to induce neutralizing responses against the vaccine strain and related laboratory-adapted, T cell-tropic HIV-1 strains, these vaccines have generally not induced neutralizing responses to primary viruses and clinical HIV-1 isolates (Hanson, 1994; Mascola et al., 1994; Matthews, 1994). This finding may be related to the general resistance of primary viruses to neutralization by sCD4 (Ashkenazi et al., 1991; Gomatos et al., 1990), monoclonal antibodies (D""Souza et al., 1995; Moore et al., 1995), and immune sera from many HIV-infected patients (Golding et al., 1994). The reason for the difference in sensitivities of primary viruses and lab isolates is not clear. It has been suggested that epigenetic factors related to the cells used to prepare the virus (Sawyer et al., 1994) and to the incorporation of host cell adhesion proteins into virion membranes (Guo and Hildreth, 1995; Hildreth and Orentas, 1989) may be involved, but it appears that structural differences in the envelope proteins of the different viruses may also be important.
Whereas it is known that some people possess potent neutralizing antibodies against primary strains of HIV, such activities are rare. Moreover, the nature of the epitopes that mediate this activity are generally unknown. A major difference between the immune responses of naturally infected individuals and those vaccinated with envelope subunit proteins is that while the humoral responses of the former are directed mostly against conformational epitopes on the viral envelope proteins that are well exposed on native virions (Moore and Ho, 1993), antibodies produced by vaccination with envelop subunit proteins are directed primarily against linear epitopes that are poorly accessible on both monomeric and cell-associated gp120 molecules (VanCott et al., 1995). The natural immune response against HIV-1 has been characterized by isolation and characterization of monoclonal antibodies (mabs) from infected individuals. These studies have utilized cell-adapted laboratory strains of HIV-1, and the mabs that have been described all have preferential neutralizing activity for lab strains over primary viruses. The major neutralization targets recognized in these studies were the V3 loop and the CD4-binding site (Chamat et al., 1992; D""Souza et al., 1994; Gorny et al., 1993; Thali et al., 1992; Tilley and Pinter, 1993).
Whereas it has been reported that some anti-V3 mabs can neutralize primary viruses (Conley et al., 1994), such neutralization is relatively inefficient, requiring 10-100 ug/ml of antibody (D""Souza et al., 1995), considerably more than that required for neutralization of susceptible lab strains. Consistent with these findings are results showing that depletion of anti-V3 antibodies from a human serum resulted in loss of neutralizing activity against the T cell-tropic MN strain, but not against several primary isolates (VanCott et al., 1995). This may be related to other evidence showing that the V3 loop in primary viruses may be buried, and not readily accessible to neutralizing antibodies (Bou-Habib et al., 1994).
A number of human mabs described in the above studies compete for binding of CD4 and have potent neutralizing activities for lab strains of HIV (Cordell et al., 1991; Ho et al., 1991; Tilley et al., 1991). These mabs are directed against conserved, conformational epitopes that are composed of residues scattered over many conserved regions of gp120 (Thali et al., 1992), including residues essential for binding of CD4 itself (Olshevsky et al., 1990). Primary viruses are much less sensitive to neutralization by these mabs than lab strains (Honnen et al., 1996; Moore et al., 1995), similar to their resistance to sCD4 itself, and there have been reports that in some cases these antibodies actually enhance infection by primary HIV-1 isolates (Lee et al., 1997; Schutten et al., 1995; Stamatatos et al., 1997). Several human mabs against other Env epitopes have been identified that have better neutralizing activities for primary isolates (Trkola et al., 1995). These include IgG b12, an anti-CD4-binding site human mab isolated from a combinatorial phage library (Burton et al., 1994), 2F5, directed against a linear epitope in gp41 (Conley et al., 1994; D""Souza et al., 1995; Muster et al., 1994; Trkola et al., 1995), and 2G12, directed against a poorly defined, glycan-dependent epitope in gp120 (Fouts et al., 1997; Trkola et al., 1996). The ability of all three of these mabs to neutralize primary viruses is a reflection of their overall increased potencies, but they also appear to have preferential activity for lab strains over primary viruses (Honnen et al., 1996).
Several studies document the role of the V1/V2 domain as a major antigenic target for HIV-1. A number of rodent mabs have been isolated from animals immunized with recombinant IIIB gp120 that are directed against linear (Fung et al., 1992) and conformational epitopes in the V2 domain (Ho et al., 1991; McKeating et al., 1993; Moore et al., 1993). HIV-infected humans have been shown to produce antibodies against linear epitopes located in both the V2 (Kayman et al., 1994; McKeating et al., 1993; Moore et al., 1993) and V1 regions (Honnen et al., 1996; Pincus et al., 1994). The linear V1 epitopes and some of the linear V2 epitopes mediate type-specific neutralization of IIIB virus and related lab strains.
Many of the anti-V2 neutralizing antibodies that have been described are directed against type-specific epitopes and appear to possess weak neutralizing activities. Thus, the significance of these antibodies for in vivo protection is unclear. Recently, however, several primate mabs have been described which have more interesting neutralizing properties. Particularly strong evidence for the role of the V1/V2 domain in neutralization of HIV-1 comes from recent studies with chimpanzee mab C108G, an antibody directed against a glycan-dependent epitope in V2 (Honnen et al., 1996; Vijh-Warrier et al., 1996; Warrier et al., 1994; Wu et al., 1995). This antibody possesses extremely potent neutralizing activities for both lab strains and primary isolates bearing the C108G epitope, including NL-HX-ADA, a primary-like, macrophage-tropic isolate.
The invention features a protein which includes a gp120 V1/V2 domain of an HIV-1 strain (or a variant or portion thereof) and not a gp120 V3 domain of an HIV-1 strain, which protein does not substantially bind CD4. For purposes of this invention, the V1/V2 domain is also intended to include the immediate conserved flanking sequences that form the conserved stem of the V1/V2 region. The gp120 V1/V2 domain of the protein displays an epitope which is recognized by an antibody which neutralizes at least one HIV-1 primary isolate with a ND90 of less than 100 xcexcg/ml. Useful V1/V2 domains include those of strain Case-A2B and strain SF162. Also included in the invention are fragments and derivatives of the V1/V2 region, including V1/V2 stem analogs in which a GAG triplet is inserted between the ends of the C1 and C2 regions (FIG. 13) and V1/V2 proteins containing deletions of various portions of the V1, V2 or the conserved flanking sequences. Such analogs can be based on any desired V1/V2 loop sequence (e.g., Case-A2B or SF162).
In various embodiments: the V1/V2 domain epitope is recognized by an antibody which neutralizes at least at least one HIV-1 primary isolate from each of at least two different clades with a ND90 of less than 100 xcexcg/ml; the two different clades are selected from the group consisting of lade A, lade B, lade C, lade D, and clade E; the V1/V2 domain epitope is recognized by an antibody which neutralizes at least two HIV-1 primary isolates of the same lade with a ND90 of less than 100 xcexcg/ml; the V1/V2 domain epitope is recognized by an antibody which neutralizes at least one HIV-1 primary isolate of at least three different clades selected from the group consisting of lade A, lade B, lade C, lade D, and lade E, with a ND90 of less than 100 xcexcg/ml; the ND90 is less than 50 xcexcg/ml; the ND90 is less than 20 xcexcg/ml; the ND90 is less than 10 xcexcg/ml; the ND90. is less than 5 xcexcg/ml; the ND90 is less than 1 xcexcg/ml; the V1/V2 domain includes a region that is at least 50%, 75%, or 90% identical to GEIKNCSFNITTSIRDKVQKEYALFY KLDIVPID; the V1/V2 domain is at least 50%, 75%, or 90% identical to VKLTPLCVTLNCIDLRNATNATSNSNTTNTTSSSGGLMMEQGEIKNCS FNITTSIRDKVQKEYALFYKLDIVPIDNPKNSTNYRLISCNTSVITQA (SEQ ID NO: 1); the protein is at least 50%, 75%, or 90% identical to LKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVTTSIRNKMQKEY ALFYKLDVVPIDNDNTSYKLINCNTSVITQACPKVS (SF162 V1/V2 loop, including 16 amino acids of the C1 flanking region, and 14 amino acids of the C2 flanking region); and the protein is a glycoprotein.
The invention also features a protein which includes a gp120 V1/V2 domain related region that is at least 50% identical to VKLTPLCVTLNCIDLRNATNATSNS NTTNTTSSSGGLMMEQGEIKNCSFNITTSIRDKVQKEYALFYKLDIVPIDNPKNS TNYRLISCNTSVITQA (SEQ ID NO: 1) and not a gp120 V3 domain of an HIV-1 strain, which protein does not substantially bind CD4, the gp120 V1/V2 domain related region displaying an epitope which is recognized by an antibody which neutralizes at least one HIV-1 primary isolate with a ND90 of less than 100 xcexcg/ml.
The invention also features a protein which includes a gp120 V1/V2 domain of an HIV-1 strain and not a gp120 V3 domain of an HIV-l strain, which protein does not substantially bind CD4. The protein, when used to immunize a rat, being capable of eliciting an antibody which neutralizes at least one HIV-1 primary isolate with a ND90 of less than 100 xcexcg/ml. In various preferred embodiments the antibody elicited neutralizes at least two HIV-1 primary isolates, at least two HIV-1 primary isolates of two different clades (e.g., clade A, clade B, clade C, clade D, and clade E).
The invention also features a monoclonal antibody which binds the gp120 V1/V2 domain of HIV-1 strain Case-A2 and neutralizes at least one HIV-1 primary isolate with a ND90 of less than 100 xcexcg/ml. In various preferred embodiments the antibody neutralizes at least two HIV-1 primary isolates, at least two HIV-1 primary isolates of two different clades (e.g., clade A, clade B, clade C, clade D, and clade E)
The invention also features a method for stimulating the formation of antibodies capable of neutralizing infection by an HIV viral isolate in at least one mammalian species, which method includes immunizing a mammalian subject with a composition comprising a protein which includes a gp120 V1/V2 domain of an HIV-1 strain and not a gp120 V3 domain of an HIV-1 strain, which protein does not substantially bind CD4. The gp120 V1/V2 domain of the protein displays an epitope which is recognized by an antibody which neutralizes at least one HIV-1 primary isolate with a ND90 of less than 100 xcexcg/ml.
In various embodiments the composition is suspended in a pharmaceutical carrier or vehicle; the composition comprises an adjuvant; such as an aluminum salt or an oil-in-water emulsion comprising a emulsifying agent and a metabolizable oil; or an immunostimulating agent and the composition is administered to the mammalian subject by injection.
It may be desirable to administer combination vaccines having one component that elicits an immune response primarily against macrophage-tropic HIV strains and a second component that elicits an immune response primarily against T Cell-tropic HIV strains. It may also be desirable for either or both components to be composed of a mixture of antigens, e.g., a mixture of antigens each of which elicits an immune response to a particular HIV strain or group of HIV strains.
The invention also includes a hybrid protein having a first part and a second part, the first part including a protein which includes a gp120 V1/V2 domain of an HIV-1 strain (or a variant thereof) and not a gp120 V3 domain of an HIV-1 strain, which protein does not substantially bind CD4, the second part including an amino terminal carrier protein comprising all or a portion of Friend MuLV gp70, preferably amino acids 1-33 or 1-263 of gp70 and, optionally, a His6 tag. The fusion protein can also include a specific cleavage site (ENLYFQS or ENLYFQG) for TEV protease (rTEV protease; Gibco, Bethesda, Md.) immediately preceding the V1/V2 region. TEV protease cleaves its specific cleavage site between the Q and S or G residues. The fusion protein can be purified on a Ni-NTA column if a His6 tag is present or by other suitable means. After digestion with TEV protease (50 U/ml at RT for 18 h) the mixture is passed over a second Ni-NTA column to remove the gp70 carrier (and TEV protease, which also carries a His6 tag). Free V1/V2 domain is recovered in the flow-through fraction.
As used herein, the term xe2x80x9ctransfected cellxe2x80x9d means any cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding a polypeptide of the invention.
As used herein, both xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d mean any chain of amino acid residues, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The polypeptides of the invention are referred to as xe2x80x9csubstantially pure,xe2x80x9d meaning that they are at least 60% by weight (dry weight) the polypeptide of interest. Preferably, the polypeptide is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, the polypeptide of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The polypeptide can be a naturally occurring, synthetic, or a recombinant molecule consisting of a hybrid with one portion, for example, encoding all or a portion of a V1/V2 domain, and a second portion being encoded by all or part of a second gene.
In the context of a polypeptide or protein, the term xe2x80x9csubstantially identical,xe2x80x9d refers to a polypeptide having a sequence that is at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% or 99% or more identical to the amino acid sequence of the reference polypeptide. For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, at least 20 amino acids, at least 25 amino acids, or preferably at least 35 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, at least 60 nucleotides, at least 75 nucleotides, or at least 90 nucleotides.
Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) with the default parameters specified therein.
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
The members of a pair of molecules (for example, an antibody-epitope pair or a receptor-ligand pair) are said to xe2x80x9cspecifically bindxe2x80x9d to each other if they bind to each other with greater affinity than to other molecules. Thus, an antibody which specifically binds to a particular epitope within a V1/V2 domain binds to that particular V1/V2 domain epitope with greater affinity than to other V1/V2 domain epitopes.
The amino acid sequences of many HIV-1 gp120 protein are described in Meyers et al. (1996).
The V1/V2 domain is that region of HIV-1 gp120 which corresponds to the following sequence from Case-A2 gp120: VKLTPLCVTLNCIDLRNATNATSNSNTTNTTSSSGGLMMEQGEIKNCSFNITTSIRD KVQKEYALFYKLDIVPI DNPKNSTNYRLISCNTSVITQA (SEQ ID NO: 1) Other V1/V2 domains can be identified by aligning SEQ ID NO: 1 with a gp120 sequence using standard sequence alignment software. Myers et al. (1996) provides alignments of a number of gp120 proteins. The four Cys residues underlined in SEQ ID NO:1 are essentially invariant and can-be used to assist in alignment. Other important highly conserved residues are the underlined Ser, Phe, Ala, and Asp residues. It should be noted that the V1/V2 domain defined above extends somewhat beyond the V1 and V2 loops as defined in Myers et al. (1996).
The xe2x80x9cV3 domainxe2x80x9d of gp120 is that region identified in Myers et al. (1996) as the V3 loop.
A protein which does not substantially bind to CD4 is a protein which does not show appreciable binding of CD4 when tested in a CD4 binding assay such as that described in U.S. Pat. No. 5,653,985.
The antigenic peptides described herein are useful in vaccine compositions or compositions used to elicit a humoral immune reponse. They may also be used in immunoassays for anti-HIV antibodies and for the production of anti-HIV antiserum.
The invention encompasses nucleic acid molecules encoding the proteins of the invention. Nucleic acid molecules within the invention can be cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded (i.e., either a sense or an antisense strand). Fragments of these molecules, which are also considered within the scope of the invention, can be produced, for example, by the polymerase chain reaction (PCR) or generated by treatment with one or more restriction endonucleases. A ribonucleic acid (RNA) molecule can be produced by in vitro transcription.
The preferred methods and materials are described below in examples which are meant to illustrate, not limit, the invention. Skilled artisans will recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing of the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.